Detection of gm crops

This presentation gives a brief overview on detection of GM CROPS.

1. Sant KABIR COLLEGE OF AGRICULTURE AND RESEARCH STATION,KAWARDHA COUSRE TITLE – Principles of seed technology COURSE NO. - APB 5221 . COURSE CREDIT- 3(1+2) YEAR :-2019-20 ASSIGNMENT ON: - Mentha SUBMITTED TO:- Shri. O.m. Narayan Verma (Assistant professor of genetics and plant breeding) SUBMITTED BY- janhavi maurya ID NO.-110518024

2. Introduction GM crops Analytical Approach Methods to detect GM crops Comparison of different methods Conclusion Content

3. Introduction Genetically modified crops  ( GM crops ) are plants used in  agriculture , the  DNA  of which has been modified using  genetic engineering  methods. In most cases, the aim is to introduce a new  trait  to the plant which does not occur naturally in the species. Examples in food crops include resistance to certain pests, diseases, environmental conditions, reduction of spoilage, resistance to chemical treatments (e.g. resistance to a  herbicide ), or improving the nutrient profile of the crop. Examples in non-food crops include production of  pharmaceutical agents ,  biofuels , and other industrially useful goods, as well as for  bioremediation . [1] Farmers have widely adopted GM technology. Acreage increased from million hectares in 1996 to 185.1 million hectares in 2016, some 12% of global cropland. As of 2016, major crop (soybean, maize, canola and cotton) traits consist of herbicide tolerance (95.9 million hectares) insect resistance (25.2 million hectares), or both (58.5 million hectares). In 2015, 53.6 million ha of GM maize were under cultivation (almost 1/3 of the maize crop). 

4. Genetically modified crops What is a GM Crop? A GM or transgenic crop is a plant that has a novel combination of genetic material obtained through the use of modern biotechnology. For example, a GM crop can contain a gene(s) that has been artificially inserted instead of the plant acquiring it through pollination. The resulting plant is said to be “genetically modified” although in reality all crops have been “genetically modified” from their original wild state by domestication, selection, and controlled breeding over long periods of time.

5. Analytical approach In general the procedure consists of three distinct steps: 1) Detection: The objective is to determine whether aproduct is GM or not. For this purpose, a generalscreening method can be used. The result is apositive/negativestatement. The screening methods areusually based on the PCR, immunoassays or bioassays.Analytical methods for detection must be sensitive andreliable enough to obtain accurate and precise results. 2) Identification: The purpose of identification is to findout which GM crop or product are present and whetherthey are authorized or not in the country. 3) Quantification: If a crop or its product has been shownto contain GM varieties, then it become necessary toassess compliance with the threshold regulation by thedetermination of the amount of each of the GM varietypresent. Normally, quantification is performed using Real-time PCR.

6. The analytical methods differ in many levels. The methods are DNA-based, protein-based or trait-based. METHODS FOR DETECTING GM CROPS AND PRODUCTS DNA-based methods DNA based methods are based on detection of thespecific genes, or DNA genetically engineered into thecrop. Although, there are several DNA based methodologies , the most commercial testing is conducted . Using PCR technology. The PCR technique is based onmultiplying a specific target DNA allowing the million or billion fold amplification by two synthetic oligonucleotide Primers . In PCR, the first step in a cycle involves . Separation of the two strands of the original DNA molecule . The second step involves binding of the two pri mers to their oligonucleotide primers. The third stepInvolves making two perfect copies of the original doublestranded DNA molecule by adding the right nucleotides tothe end of each primer, using the strands as templates.Once the cycle is completed, it can be repeated, and foreach cycle the number of copies is doubled, resulting inan exponential plified cation. The amplified fragment can Be detected by gel electrophoresis or hybridization techniques.

7. . Qualitative PCR analysis The most critical parameter for successful PCR is the design of primers. A poorly designed primer can result in little or no product due to non-specific amplification and/or primer-dimer formation, which can become competitive enough to suppress product formation. It is essential that care should be taken in the design of primers for PCR. Several parameters including the length of the primer, %GC content and the 3' sequence need to be optimized for successful PCR. Certain of these parameters can be manually optimized while others are best done with computer programs. The s trategies for choosing an appropriate target are as follows: The detection of GM crops: For general screening purposes the focus should be on target sequences that are characteristic for the group to be screened (Figure1A). Genetic control elementssuch as the cauliflower mosaic virus 35S promoter (P-35S) and theAgrobacterium tumefaciens nos terminator (nos3’) arepresent in many GM crops currently on the market

8. III. Quantitative PCR In principle, PCR based quantitation can be performedeither after completion of the PCR (end-point analysis), or during the PCR (real-time analysis). Quantification using conventional PCR: Conventional PCR measures the products of the PCR reaction at the end point in the reaction profile. End-point analyses are based on comparison of the final amount of amplified DNA of two DNA targets, the one to be quantified and a competitor (an artificially constructed DNA that is addedin a small and known quantity prior to the PCR fication ation and that is co-amplified with the target,which is to be quantified). Quantification using Real-time PCR: Real-time PCR is a system based on the continuous monitoring of PCR products. This is done via fluorometric measurement of an internal probe during the reaction. In real-time analyses the amount of product synthesized during PCR is estimated directly by measurement of fluorescence in the PCR reaction. Several types of hybridisation probes are available that will emit fluorescent light correspondingto the amount of synthesized DNA. However, the amount of synthesised product can also be estimated with fluorescent dyes, e.g. SYBR Green I that intercalates double-stranded DNA. I. Multiplex PCR-based detection methods With multiplex PCR-based methods, several target DNAsequences can be screened and detected in a singlereaction. The advantage of multiplex methods is evidentlythat fewer reactions are needed to test a sample forpotential presence of GMO-derived DNA. Development ofmultiplex assays requires careful testing and validation.After the PCR the resulting pool of amplified fragments needs to be further analysed to distinguish between thevarious amplicons.

9. Protein based methods Immunoassay is the current method for detection and quantification of new (foreign) proteins introduced through genetic transformation of plants. Immunoassay is based on the specific binding between an antigen and an antibody. Thus, the availability of antibodies with the desired affinity and specificity is the most important factor for setting up immunoassay systems. I. ELISA (Enzyme Linked Immunosorbent Assay) In ELISA the antigen-antibody reaction takes place on asolid phase (microtiter plates). Antigen and antibody react and produce a stable complex, which can be visualised by addition of a second antibody linked to an enzyme.Addition of a substrate for that enzyme results in a colourformation, which can be measured photometrically orrecognised by naked eye.ELISA test kits provide the quantitative results in hourswith detection limits less than 0.1%. However, somecompanies operate with slightly higher quantification ls vels as e.g. 0.3%. ELISAs have been designed to detect a novel GM protein or trait. II. Lateral flow sticks The lateral flow test (dipstick format) uses a membrane-based detection system. The membrane contains twocapture zones, one captures the bound GM protein, the otherr captures color reagent. Paper strips or plasticpaddles are used as support for the capture antibody that is immobilized onto a test strip in specific zone. Most Tests are provided usually in kit form.

10. Comparison of the different methods The comparison of various detection methods is summarized in Table 1. At present, only PCR offers away for performing a general screening for GM varieties and detection of particular “events”. Phenotypic characterisation and immunoassays detect particular traits that may be present in several GM crops (e.g. The Cry1a protein and genes, conferring insecticide resistance , are present in a range of different GM Maize: MON80100, MON801, MON802, MON809, 176, BT11). One of the major considerations in Analyticall testing of almost any GM crop or its product is the sampling procedure. . The sample analysed must be representative of the material from which it is mple n otherwise the testing regime is flawed. Sample preparation for both DNA-based and protein-based methods is critical for detection and /or quantification. It is important to know the limitations of each procedure as well as the purpose of detection. Both the sample size and sampling procedures dramatically impact the conclusions that may be drawn from any of these testing methods.

11. CONCLUSIONS The release of GM crop and products in environment andmarkets worldwide has resulted in public debate in manypart of the world. Despite the continuing controversyabout GM crops, the hectarage and number of farmersg g rowing GM crops have continued to grow at a double digit rate or more, every year. Currently, there arefourteen countries, 9 developing countries and 5 developed countries, growing GM crops. The need for identification and detection of GM crops and products has increased with the rapid expansion in the cultivation of GM crops. Labeling and traceability of GM material isway forward to address the concerns of consumers and regulators. The establishment of relevant, reliable and onomical al methodology for detection, identification and on antification of GM crops continues to be a challenge atinternational level. A great number of different strategies international are available for testing of GM material and the quality of these results depends not only on the methodology and the equipment but also the sampling, the theoretical expertise and the practical skills of the regulatory officers handling the testing of the sample. Therefore , it is important to understand the methods and the ir applications for detection of GM crops and theirproducts.

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